Required to isolate the bacteria from the clinical specimens CONSTITUENTS OF CULTURE MEDIA Constituent Explanation 1Water Distilled water or potable water with low mineral content Serves as the source of hydrogen and oxygen ID: 1039095
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1. CULTURE MEDIA
2. CULTURE MEDIARequired to isolate the bacteria from the clinical specimens. CONSTITUENTS OF CULTURE MEDIAConstituentExplanation1)WaterDistilled water or potable water with low mineral content.Serves as the source of hydrogen and oxygen. 2)ElectrolytesSodium chloride or other electrolytes.
3. CONSTITUENTS OF CULTURE MEDIAPeptone: Complex mixture of partially digested proteins. Source- from lean meat or other protein material such as heart muscle, casein or fibrin, or soya flour usually by digestion with proteolytic enzymes such as pepsin.Constituents - proteoses, aminoacids, inorganic salts (phosphates, potassium magnesium) and accessory growth factors like nicotinic acid and riboflavin.
4. CONSTITUENTS OF CULTURE MEDIA AgarUsed for solidifying agent.Agar (agar-agar) is prepared from the cell wall of variety of seaweeds. Components- Long-chain polysaccharide (D-galctopyranose units) and a small amount of protein-like material, long chain fatty acids and traces of inorganic salts (calcium and magnesium).Agar is preferred over gelatineBacteriologically inertMelts at 98°C and usually solidifies at 42°C.Does not add any nutritive property to the culture medium. Whereas gelatin is liquefied by a number of bacteria and it melts at 24°C, and remains in liquid state at room temperature.
5. CONSTITUENTS OF CULTURE MEDIA AgarConcentration of agar used: Solid agar preparation - 1-2% Semisolid agar- 0.5%Solid agar to inhibit Proteus swarming- 6%Preparation of agar media- The appropriate amount of agar powder is added to water and the mixture is dissolved and sterilized by placing it in an autoclave. When the temperature of the molten agar comes down to 450C, it is poured to the petri dishes and then allowed to set for 20 minutes.
6. CONSTITUENTS OF CULTURE MEDIA Meat extractHighly concentrated meat stock, usually made from beef, contains protein degradation products, inorganic salts, carbohydrates and growth factors. Yeast extract Prepared from washed cells of Baker’s yeast. It contains aminoacids, inorganic salts (potassium and phosphates) and carbohydrates.Malt extract-Consists of maltose (about 50%), starch, dextrin, glucose and 5% protein products.
7. CONSTITUENTS OF CULTURE MEDIA Blood and serumImportant components of enriched media and provide extra nutrition to fastidious bacteria. Usually 5-10% of sheep blood is used. Horse, ox or human blood also can be used. Blood should be collected aseptically and rendered non-coagulable by defibrillation (by shaking the blood in a bottle containing sterile glass beads) or adding oxalate or citrate. Serum is sterilized by filtration after collection.
8. TYPES OF CULTURE MEDIA Routine laboratory media-They are prepared from nutrients such as aqueous extract of meat, peptone etc. Simple/ basal media Enriched media Enrichment brothSelective mediaDifferential mediaTransport mediaAnaerobic mediaDefined or synthetic mediaSimple synthetic mediaComplex synthetic mediaBased on consistency, culture media are grouped into:Liquid media (or broth)Semisolid mediaSolid media2. Based on growth requirements, culture media are classified as-
9. Simple/ basal mediaContain minimum ingredients that support the growth of non-fastidious bacteria. Peptone water-It contains peptone (1%) + NaCl (0.5%) + water Nutrient broth- It is made up of peptone water + meat extract (1%). Nutrient agar-It is made up of nutrient broth + 2% agar. Semisolid medium: It is prepared by reducing the concentration of agar to 0.2–0.5 %.
10. Uses of basal mediaTesting the non-fastidiousness of bacteriaThey serve as the base for the preparation of many other media.Nutrient broth is used for studying the bacterial growth curveNutrient agar is the preferred medium for-Performing the biochemical tests such as oxidase, catalase and slide agglutination test etc.To study the colony characterPigment demonstration
11. Enriched MediaBlood agar - Prepared by adding 5-10% of sheep blood to the molten nutrient agar at 450C.Tests the hemolytic property of the bacteria, which may be either- i) partial or α (green) hemolysis and ii) complete or β hemolysisBasal medium is added with additional nutrients such as blood, serum or egg. In addition to non-fastidious organisms, they also support the growth of fastidious nutritionally exacting bacteria.
12. Enriched Media More nutritious than blood agar, and even supports certain highly fastidious bacteria such as Haemophilus influenzae that does not grow on blood agar.Chocolate agarHeated blood agar, prepared by adding 5-10% of sheep blood to the molten nutrient agar at 700C
13. Enriched MediaBlood culture media- Culturing microorganisms from blood specimen. They are either monophasic or biphasic media. Monophasic medium -brain heart infusion (BHI) broth.Biphasic medium has a liquid phase containing BHI broth and a solid agar slope made up of BHI agar.Loeffler's serum slope- is used for isolation of Corynebacterium diphtheriae.
14. Enrichment brothLiquid media added with some inhibitory agents which selectively allow certain organism to grow and inhibit others.Important for isolation of the pathogens from clinical specimens which also contain normal flora (e.g. stool and sputum specimen). Tetrathionate broth (used for Salmonella Typhi)Gram negative broth- for isolation of ShigellaSelenite F broth–for isolation of ShigellaAlkaline peptone water (APW)- for Vibrio cholerae
15. Selective mediaMediaUsed for isolation ofLowenstein Jensen (LJ) mediumMycobacterium tuberculosis Thiosulphate Citrate Bile salt Sucrose (TCBS)Vibrio species DCA (Deoxycholate Citrate Agar) Salmonella and Shigella from stool XLD (Xylose Lysine Deoxycholate) agarSalmonella and Shigella from stool Potassium tellurite agar (PTA)Corynebacterium diphtheriaeWilson Blair bismuth sulphite mediumSalmonella Typhi.Solid media containing inhibitory substances that inhibit the normal flora present in the specimen and allow the pathogens to grow.
16. Selective media Lowenstein LJ Media
17. Transport mediaUsed for the transport of the clinical specimens suspected to contain delicate organism or when the delay is expected while transporting the specimens. Bacteria do not multiply, they only remain viable.OrganismTransport mediaStreptococcusPike’s mediumNeisseriaAmies medium, Stuart’s mediumVibrio choleraeVR (Venkatraman-Ramakrishnan) mediumAutoclaved sea waterCary Blair mediumShigella, SalmonellaBuffered glycerol salineCary Blair medium
18. Differential media Differentiate between two groups of bacteria by using an indicator.Differential media Features1)MacConkey agarDifferential and low selective medium -used for the isolation of enteric GNB. Differentiates organisms into LF (pink colonies, e.g. Escherichia coli) and NLF or (colorless colonies, e.g. Shigella). Composition- Peptone, lactose, agar, neutral red (indicator) and taurocholate
19. Differential media Differential media Features2)CLED agar -Cysteine lactose electrolyte-deficient agarSimilar to MacConkey agar.Used as an alternative to combination of blood agar and MacConkey agar, for the processing of urine specimensAdvantages over MacConkey agar-It is less inhibitory than MacConkey agar, supports the growth of Gram positive bacteria (except β hemolytic Streptococcus) and Candida.Advantage over blood agar -It can prevent the swarming of Proteus.
20. Anaerobic Culture MediaContain reducing substances which take-up oxygen and create lower redox potential and thus permit the growth of obligate anaerobes such as Clostridium. Robertson’s cooked meat (RCM) brothContains chopped meat particles (beef heart), which provide glutathione (a sulfhydryl group containing reducing substance) and unsaturated fatty acids. Most widely used anaerobic culture medium.Also used for maintenance of stock cultures.
21. Anaerobic Media
22. Anaerobic Culture Media (cont..)Other anaerobic media includeThioglycollate broth Anaerobic blood agarBHIS agar-Brain heart infusion agar with supplements (vitamin K and hemin)Neomycin blood agarEgg yolk agarPhenyl ethyl agarBacteroides bile esculin agar (BBE agar)
23. Synthetic mediaChemically defined media are used for various experimental purposes, prepared exclusively from pure chemical substances and their composition i.e exact quantity of each chemical used is known.
24. Simple synthetic media Provide the basic essentials for the growth of many non-fastidious heterotrophs but they will not support growth of fastidious bacteria.
25. Complex synthetic media - In addition to the above, certain aminoacids, purines, pyrimidines, and other growth factors are incorporated. Hence, they can also support the growth of more exacting bacteria.
26. References Textbook of Medical Microbiology by Ananthnarayan, PanikerTextbook of Medical Microbiology by C.P Baweja Textbook of Medical Microbiology by S. Bhat, A.S.SastryTextbook of Medical Microbiology by D.R.Arora, Brij bala Arora